Seven gliotoxin-related compounds were isolated from the fungus Penicillium sp. strain JMF034, obtained from deep sea sediments of Suruga Bay, Japan. These included two new metabolites, bis(dethio)-10a-methylthio-3a-deoxy-3,3a-didehydrogliotoxin (1) and 6-deoxy-5a,6-didehydrogliotoxin (2), and five known metabolites (3−7). The structures of the new compounds were elucidated by analysis of spectroscopic data and the application of the modified Mosher's analysis. All of the compounds exhibited cytotoxic activity, whereas compounds containing a disulfide bond showed potent inhibitory activity against histone methyltransferase (HMT) G9a. None of them inhibited HMT SET7/9. A fter intensive investigation of secondary metabolites of marine macroorganisms for almost half a century, 1 some marine natural product chemists concluded that new structural entities from these organisms were nearly exhausted. Therefore, they turned their attention to marine microorganisms as an untapped source of secondary metabolites. 2,3 With this trend in mind we have been searching for cytotoxic metabolites produced by marine-derived fungi. We found that a Penicillium sp. (strain JMF034) isolated from deep-sea sediments collected in Suruga Bay exhibited potent cytotoxic activity. From the culture medium we isolated a series of metabolites related to gliotoxin 4 and gliotoxin itself as the active constituents, among which two metabolites are new. Gliotoxin is a representative member of the epipolythiodioxopiperazine (ETP) class of fungal metabolites.5 Due to its potent cytotoxicity toward cancer cell lines, this compound has been considered as a lead for anticancer agents.5 Recently, dimeric ETPs were shown to inhibit histone methyltransferase (HMT). 6 These observations prompted us to examine the HMT-inhibitory activity of the metabolites, some of which showed significant activity. Here we describe structure elucidation of the new compounds.The culture medium of Penicillium sp. JMF034 was extracted with EtOAc, and the combined EtOAc layers were fractionated by solvent partitioning, gel permeation, and reversed-phase column chromatographies followed by ODS-HPLC to yield compounds 1−7. The known compounds were identified as bis(dethio)bis(methylthio)gliotoxin (3), 7 bis(dethio)bis-(methylthio)-5a,6-didehydrogliotoxin (4), 8 5a,6-didehydrogliotoxin (5), 9 gliotoxin (6), 10 and gliotoxin G (7), 8 on the basis of ESIMS and 1 H NMR data.The molecular formula of compound 1 was determined to be C 14 H 16 N 2 O 3 S on the basis of the HRESIMS data. Analysis of the 1 H NMR spectrum (Table 1) in conjunction with the HSQC data revealed two singlet methyls, a pair of nonequivalent methylene protons, two oxygen-or nitrogensubstituted methines, five olefinic protons, two of which were a pair of exomethylene protons, and an exchangeable proton. Even though the number of protons was small, interpretation of the COSY spectrum was complex due to the overlap of H-5a and H-6, the absence of coupling between H-6 and H-7, and the observation of long-ran...

SET domain containing lysine methyltransferase 7/9 (Set7/9), a histone lysine methyltransferase (HMT), also methylates non-histone proteins including estrogen receptor (ER) α. ERα methylation by Set7/9 stabilizes ERα and activates its transcriptional activities, which are involved in the carcinogenesis of breast cancer. We identified cyproheptadine, a clinically approved antiallergy drug, as a Set7/9 inhibitor in a high-throughput screen using a fluorogenic substrate-based HMT assay. Kinetic and X-ray crystallographic analyses revealed that cyproheptadine binds in the substrate-binding pocket of Set7/9 and inhibits its enzymatic activity by competing with the methyl group acceptor. Treatment of human breast cancer cells (MCF7 cells) with cyproheptadine decreased the expression and transcriptional activity of ERα, thereby inhibiting estrogen-dependent cell growth. Our findings suggest that cyproheptadine can be repurposed for breast cancer treatment or used as a starting point for the discovery of an anti-hormone breast cancer drug through lead optimization.

Vitamin D3 metabolites inhibit the expression of lipogenic genes by impairing sterol regulatory element-binding protein (SREBP), a master transcription factor of lipogenesis, independent of their canonical activity through a vitamin D receptor (VDR). Herein, we designed and synthesized a series of vitamin D derivatives to search for a drug-like small molecule that suppresses the SREBP-induced lipogenesis without affecting the VDR-controlled calcium homeostasis in vivo. Evaluation of the derivatives in cultured cells and mice led to the discovery of VDR-silent SREBP inhibitors and to the development of KK-052 (50), the first vitamin D-based SREBP inhibitor that has been demonstrated to mitigate hepatic lipid accumulation without calcemic action in mice. KK-052 maintained the ability of 25-hydroxyvitamin D3 to induce the degradation of SREBP but lacked in the VDR-mediated activity. KK-052 serves as a valuable compound for interrogating SREBP/SCAP in vivo and may represent an unprecedented translational opportunity of synthetic vitamin D analogues.

Background:The spatiotemporal regulation of Rac1 controls cell migration. Results: EGF induced two waves of Rac1 activation in the process of cell migration. Conclusion: 14-3-3 proteins regulate the second EGF-induced wave of Rac1 activation by interacting with RacGEF. Significance: The second wave of Rac1 activation might be required for EGF-induced cell migration.

In a previous study, we reported a novel 14-membered ring macrolide, migrastatin isolated from culture broth of Streptomyces sp. MK929-43F1 as an inhibitor of tumor cell migration1). Recently, however, we found that our original migrastatin isolate contained teleocidin-related compounds which display strong inhibitory activity against tumor cell migration. In this report, we describe the effects of teleocidin-free migrastatin on tumor cell migration and on the growth of several types of tumor cells. Whenthe original migrastatin sample was subjected to HPLC (Capcell Pak C18 column, 20X250mm) developing with 70% aq acetonitrile, a potent migration inhibitory activity was eluted in a fraction other than migrastatin, indicating that the migration inhibitory activity of our original migrastatin samples reported previously was due to the activity of an impurity. The potent migration inhibitory fraction contained teleocidin-related compounds, as judged from UV spectrum. Teleocidin-related compounds are knownto inhibit [3H]PDBu binding to the cell surface2), therefore, we clarified the content of teleocidin-related compoundsin the original migrastatin sample as evaluated by [3H]PDBu binding assay (Fig. 1) migrastatin reported previously4), possibly due to the low content of impurities. Migration inhibitory activities of pure migrastatin and pendolmycin were assayed by the wound healing method as described before1^In brief, a standardized scratch was madethrough a con fluent monolayer of humanesophageal carcinoma EC17cells, and then the cells from the cut edge were allowed to migrate for 24 hours5). Pendolmycin as well as teleocidin B inhibited migration of EC17 cells at lOng/ml (data not shown). On the other hand, EC17 cells migrated inwardly and covered a great area of the scratch even in the presence of 100 /ig/ml of migrastatin. However, when the EC17 cells were pretreated with migrastatin

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Histone lysine methyltransferases (HMTs) regulate transcriptional activity by writing epigenetic marks. Methylation of histone H3K9, a hallmark of silent chromatin, 1 is mainly regulated by two subgroups of HMTs, G9a/G9a-like protein 2 and Suv39h. 3 G9a and G9a-like protein induce mono-and di-methylation of histone H3K9 (H3K9me1 and H3K9me2) in the euchromatin region, 4 whereas Suv39h contributes to tri-methylation of histone H3K9 (H3K9me3) in the heterochromatin. 3 As methylation at histone H3K9, increased DNA methylation and reduced levels of activating chromatin modifications (e.g., histone acetylation) have been detected at promoter regions of aberrantly silenced tumor suppressor genes in cancer cells, 5,6 HMTs responsible for histone H3K9 methylation may represent promising targets for drug discovery. Indeed, overexpression of G9a is associated with several types of cancers and downregulation of G9a by RNAi inhibits tumor cell proliferation. 7-9 Chaetocin and BIX-01294 are small molecules that inhibit histone H3K9 HMTs. Chaetocin, which inhibit both G9a and Suv39h activities, is a member of the epipolythiodioxopiperazine (ETP) class of fungal metabolites. 10 On the other hand, BIX-01294 is a synthetic compound that selectively inhibits G9a but not Suv39h1 11 Based on a co-crystallization analysis of G9a with BIX-01294, BIX-01294-related molecules have been developed; these novel compounds are both more potent inhibitors and more membrane permeable than the parent compound. 12-15 MATERIALS AND METHODS MaterialsAnti-mono-and di-methylated histone H3K9 antibodies were purchased from Merck Millipore (Billerica, MA, USA). pGEX4T-1-mG9a (706Àstop amino acid (a.a.)), pGEX4T-3-histone H3 (1À57 a.a.) and pGEX4T-3-mSuv39h1-H320R (74À412 a.a.) were previously described. 2 The pET-28a(+)-Set7/9 was kindly provided by Dr Kenichi Nishioka (National Institute of Genetics). Bacterial protein expression and purificationpGEX4T-1-mG9a (706Àstop a.a.), pGEX4T-3-mSuv39h1-H320R (74À412 a.a.), pET-28a(+)-Set7/9, or pGEX4T-3-histone H3 (1À57 a.a.) was introduced into Escherichia coli BL21 (DE3). Expression of each recombinant protein was induced by 0.1 mM isopropyl-b-D-galactopyranosid at 18 1C for 24 h. Purification of GST-and (His) 6 -fused proteins were carried out by either glutathioneaffinity (GE Healthcare UK Ltd, Little Chalfont, UK) or nickel (Ni 2+ ) affinity (GE Healthcare UK Ltd). In vitro HMT assayEach purified recombinant HMT was pretreated in the presence or absence of a given compound in HMT buffer (50 mM Tris-HCl (pH 8.5), 10 mM MgCl 2 , 20 mM KCl, 10 mM 2-mercaptoethanol and 250 mM sucrose) containing 3 mg ml À1 of BSA for 1 h. Next, 10 mg ml À1 of SAM and 20 mg ml À1 of GST-fused histone H3 (1À57 a.a.) were added into the reaction mixture and further incubated for 1 h at 37 1C. To detect histone methylation on western blots, samples were transferred onto Immobilon membranes (Merck Millipore), probed using appropriate primary antibodies, and visualized using horseradish peroxidase-linked secondary antibodies.In or...

Cell migration is an essential step for tumor metastasis. The small GTPase Rac1 plays an important role in cell migration. Previously, we reported that epidermal growth factor (EGF) induced two waves of Rac1 activation; namely, at 5 min and 12 h after stimulation. A second wave of EGF-induced Rac1 activation was required for EGF-induced cell migration, however, the spatiotemporal regulation of the second wave of EGF-induced Rac1 activation remains largely unclear. In this study, we found that 5-lipoxygenase (5-LOX) is activated in the process of EGF-induced cell migration, and that leukotriene C4 (LTC4) produced by 5-LOX mediated the second wave of Rac1 activation, as well as cell migration. Furthermore, these effects caused by LTC4 were found to be blocked in the presence of the antagonist of cysteinyl leukotriene receptor 1 (CysLT1). This blockage indicates that LTC4-mediated CysLT1 signaling regulates the second EGF-induced wave of Rac1 activation. We also found that 5-LOX inhibitors, CysLT1 antagonists and the knockdown of CysLT1 inhibited EGF-induced T cell lymphoma invasion and metastasis-inducing protein 1 (Tiam1) expression. Tiam1 expression is required for the second wave of EGF-induced Rac1 activation in A431 cells. Therefore, our results indicate that the 5-LOX/LTC4/CysLT1 signaling pathway regulates EGF-induced cell migration by increasing Tiam1 expression, leading to a second wave of Rac1 activation. Thus, CysLT1 may serve as a new molecular target for antimetastatic therapy. In addition, the CysLT1 antagonist, montelukast, which is used clinically for allergy treatment, might have great potential as a novel type of antimetastatic agent.